Myocilin is the gene product most closely linked to early-onset, inherited primary open angle glaucoma (POAG), accounting for more than 10% of juvenile and 5% of adult-onset disease. (Kwon Y H, Fingert J H, Kuehn M H, & Alward W L (2009) Primary open-angle glaucoma. N. Engl. J. Med. 360(11):1113-1124; Fingert J H, et al. (1999) Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum Mol Genet 8(5):899-905) Even though it is expressed throughout the body, myocilin only appears to cause disease as part of its role in the trabecular meshwork (TM), an extracellular matrix in the anterior segment of the eye that controls aqueous humor outflow and is involved in regulating intraocular pressure (IOP). (Resch Z T & Fautsch M P (2009) Glaucoma-associated myocilin: a better understanding but much more to learn. Exp Eye Res 88(4):704-712) Dysregulation of fluid flow leads to elevated IOP, a major risk factor for glaucoma. (Alward W L (1998) Medical management of glaucoma. N. Engl. J. Med. 339(18):1298-1307) Amino acid altering mutations in the gene encoding myocilin lead to sequestration and accumulation of mutant myocilin, particularly in the ER of TM cells with toxic consequences of cell stress and death that lead to a compromised TM and a hastening of glaucoma phenotypes. (Joe M K, et al. (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem Biophys Res Commun 312(3):592-600; Liu Y & Vollrath D (2004) Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum Mol Genet 13(11):1193-1204; Yam G H-F, Gaplovska-Kysela K, Zuber C, & Roth J (2007) Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma. Am. J. Pathol. 170(1):100-109; Gobeil S, Letartre L, & Raymond V (2006) Functional analysis of the glaucoma causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion. Exp Eye Res 82(6):1017-1029; Vollrath D & Liu Y (2006) Temperature sensitive secretion of mutant myocilins. Exp Eye Res 82(6):1030-1036; Wang L, et al. (2007) Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells. Mol Vis 13:618-625)
Interestingly, over 70 mutations in myocilin, clustered in its C-terminal ˜30 kDa olfactomedin (OLF) domain, have been documented, with differing severity in terms of age of onset, cellular toxicity, and extent of thermal destabilization of the OLF domain. (Gobeil S, Letartre L, & Raymond V (2006) Functional analysis of the glaucoma causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion. Exp Eye Res 82(6):1017-1029; Vollrath D & Liu Y (2006) Temperature sensitive secretion of mutant myocilins. Exp Eye Res 82(6):1030-1036; Gong G, Kosoko-Lasaki O, Haynatzki G R, & Wilson M R (2004) Genetic dissection of myocilin glaucoma. Hum. Mol. Genet. 13:R91-102; Burns J N, et al. (2010) Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones. ACS Chem Biol 5(5):477-487; Burns J N, Turnage K C, Walker C A, & Lieberman R L (2011) The stability of myocilin olfactomedin domain variants provides new insight into glaucoma as a protein misfolding disorder. Biochemistry 50(26):5824-5833)
Importantly, pathogenesis is a gain-of-toxic-function, as myocilin knockout mice, and individuals harboring premature stop codons that prevent myocilin translation, do not develop glaucoma. (Gould D B, et al. (2006) Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma. Mol Cell Biol 26(22):8427-8436; Lam D S, et al. (2000) Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma. Invest Ophthalmol Vis Sci 41(6):1386-1391) The nature of the aggregate and its toxicity has not been unambiguously identified, but the Unfolded Protein Response is upregulated in cells expressing high levels of wild-type myocilin and ER stress response genes are upregulated both in cells and in mice expressing mutant myocilin. (Joe M K, et al. (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem Biophys Res Commun 312(3):592-600; Carbone M A, et al. (2009) Overexpression of myocilin in the Drosophila eye activates the unfolded protein response: implications for glaucoma. PLoS One 4(1):e4216; Zode G S, et al. (2011) Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest 121(9):3542-3553)
Mutant myocilin also readily forms a detergent-insoluble species consisting of amyloid fibrils, a specific misfolded species that is recalcitrant to disaggregation, in vitro and in a cellular model. (Zhou Z & Vollrath D (1999) A cellular assay distinguishes normal and mutant TIGR/myocilin protein. Hum Mol Genet 8(12):2221-2228; Orwig S D, et al. (2011) Amyloid Fibril Formation by the Glaucoma-Associated Olfactomedin Domain of Myocilin. J Mol Biol)
In spite of the interest in developing therapeutic routes to mitigate myocilin aggregation and toxicity, primarily by promoting its secretion, it is not understood why myocilin, unlike other mutant proteins, is not efficiently cleared by ER-associated degradation (ERAD). (Liu Y & Vollrath D (2004) Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum Mol Genet 13(11):1193-1204; Burns J N, et al. (2010) Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones. ACS Chem Biol 5(5):477-487; Zode G S, et al. (2011) Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest 121(9):3542-3553; Jia L Y, et al. (2009) Correction of the disease phenotype of myocilin-causing glaucoma by a natural osmolyte. Invest Ophthalmol Vis Sci 50(8):3743-3749; Yam G H, Gaplovska-Kysela K, Zuber C, & Roth J (2007) Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 48(4):1683-1690; Zode G S, et al. (2012) Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 53(3):1557-1565) Misfolded proteins are typically efficiently ubiquitinated in the ER and retro-translocated to the cytosol for proteasomal degradation, a mechanism that appears to be challenged in the case of mutant myocilin.
Chaperone 4 proteins within the ER, primarily ATPases Grp94 (an Hsp90 family member) and Grp78 (an Hsp70 member, also called BiP), are essential for triage decisions about protein fate. (Meusser B, Hirsch C, Jarosch E, & Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7(8):766-772) The exact order in which ER clients are processed by chaperones is unknown; however Grp94 seems to be much more selective for a distinct client sub-set. (Eletto D, Dersh D, & Argon Y (2010) GRP94 in ER quality control and stress responses. Semin Cell Dev Biol 21(5):479-485) Indeed, Grp94 and Grp78 have been shown to co-localize with mutant myocilin, but the significance of this co-localization has remained elusive. ERAD-related loss of function due to inherited mutation is associated with myriad diseases such as cystic fibrosis and Gaucher disease, among many others. (Joe M K, et al. (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem Biophys Res Commun 312(3):592-600; Liu Y & Vollrath D (2004) Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum Mol Genet 13(11):1193-1204; Zode G S, et al. (2011) Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest 121(9):3542-3553; Yam G H, Gaplovska-Kysela K, Zuber C, & Roth J (2007) Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 48(4):1683-1690; Farinha C M & Amaral M D (2005) Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol Cell Biol 25(12):5242-5252; Ron I & Horowitz M (2005) ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity. Hum Mol Genet 14(16):2387-2398) A better understanding of mutant myocilin ER retention could lead to corrective measures that would reduce its accumulation through manipulation of the ER quality control system.
The hereditary form of open angle glaucoma is linked to missense mutations in the MYOC gene. Mutant myocilin misfolding and aggregation in trabecular meshwork cells causes a toxic gain-of-function, namely, cell death, which hastens an increase in intraocular pressure, a primary risk factor for glaucoma. Since the absence of myocilin has no obvious consequence in humans or mice, the inventors speculated that developing ways to deplete aberrant myocilin could be clinically relevant for glaucoma.